RESUMO
MicroRNAs (miRNAs) are short RNAs with essential roles in gene regulation in various organisms including higher plants. In contrast to the vast information on miRNAs from many economically important plants, almost nothing has been reported on the identification or analysis of miRNAs from rubber tree (Hevea brasiliensis L.), the most important natural rubber-producing crop. To identify miRNAs and their target genes in rubber tree, high-throughput sequencing combined with a computational approach was performed. Four small RNA libraries were constructed for deep sequencing from mature and young leaves of two rubber tree clones, PB 260 and PB 217, which provide high and low latex yield, respectively. 115 miRNAs belonging to 56 known miRNA families were identified, and northern hybridization validated miRNA expression and revealed developmental stage-dependent and clone-specific expression for some miRNAs. We took advantage of the newly released rubber tree genome assembly and predicted 20 novel miRNAs. Further, computational analysis uncovered potential targets of the known and novel miRNAs. Predicted target genes included not only transcription factors but also genes involved in various biological processes including stress responses, primary and secondary metabolism, and signal transduction. In particular, genes with roles in rubber biosynthesis are predicted targets of miRNAs. This study provides a basic catalog of miRNAs and their targets in rubber tree to facilitate future improvement and exploitation of rubber tree.
Assuntos
Genoma de Planta/genética , Estudo de Associação Genômica Ampla/métodos , Hevea/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , MicroRNAs/genética , Quitinases/genética , Quitinases/metabolismo , Biblioteca Gênica , Hevea/metabolismo , MicroRNAs/isolamento & purificação , MicroRNAs/metabolismo , Muramidase/genética , Muramidase/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA de Plantas/genética , RNA de Plantas/isolamento & purificação , RNA de Plantas/metabolismo , Análise de Sequência de RNARESUMO
Natural rubber is synthesized in laticifers in the inner liber of the rubber tree (Hevea brasiliensis). Upon bark tapping, the latex is expelled due to liber turgor pressure. The mature laticifers are devoid of plasmodesmata; therefore a corresponding decrease in the total latex solid content is likely to occur due to water influx inside the laticifers. Auxins and ethylene used as efficient yield stimulants in mature untapped rubber trees, but, bark treatments with abscisic acid (ABA) and salicylic acid (SA) could also induce a transient increase latex yield. We recently reported that there are three aquaporin genes, HbPIP2;1, HbTIP1;1 and HbPIP1;1, that are regulated differentially after ethylene bark treatment. HbPIP2;1 was up-regulated in both the laticifers and the inner liber tissues, whereas HbTIP1;1 was up-regulated in the latex cells, but very markedly down-regulated in the inner liber tissues. Conversely, HbPIP1;1 was down-regulated in both tissues. In the present study, HbPIP2;1 and HbTIP1;1 showed a similar expression in response to auxin, ABA and SA, as seen in ethylene stimulation, while HbPIP1;1 was slightly regulated by auxin, but neither by ABA nor SA. The analysis of the HbPIP1;1 promoter region indicated the presence of only ethylene and auxin responsive elements. In addition, the poor efficiency of this HbPIP1;1 in increasing plasmalemma water conductance was confirmed in Xenopus oocytes. Thus, an increase in latex yield in response to all of these hormones was proposed to be the major function of aquaporins, HbPIP2;1 and HbTIP1;1. This study emphasized that the circulation of water between the laticifers and their surrounding tissues that result in latex dilution, as well as the probable maintenance of the liber tissues turgor pressure, favor the prolongation of latex flow.
Assuntos
Regulação da Expressão Gênica de Plantas , Hevea/efeitos dos fármacos , Hevea/metabolismo , Látex/metabolismo , Casca de Planta/efeitos dos fármacos , Casca de Planta/metabolismo , Aquaporinas/genética , Proteínas de Plantas/genética , Reação em Cadeia da PolimeraseRESUMO
Quebrachitol is a cyclic polyol and, along with sucrose, is one of the main sugars in Hevea latex. However, in contrast to sucrose, the mechanism and regulation of quebrachitol absorption is still unknown. Screening a latex-derived cDNA library using polyol transporter-specific probes, two full-length cDNAs were isolated, and named HbPLT1 and HbPLT2 (for Hevea brasiliensis polyol transporter 1 and 2, respectively). Their respective sequences exhibited close similarity with the previously cloned acyclic sugar polyol transporters, and shared the main features of the major facilitative superfamily. The functional activity of one of the cDNAs was determined by using an HbPLT2-complemented yeast strain. These strains displayed a marginal absorption of cyclic (inositol) and acyclic (mannitol and sorbitol) polyol but no absorption of sucrose, hexose and glycerol. Active absorption for xylitol was detected, and was competitively inhibited by quebrachitol. HbPLT1 and HbPLT2 expression patterns varied in response to different stimuli. Bark treatment with ethylene resulted in an early and significant up-regulation of HbPLT2 transcripts in laticifers as well as in inner bark cells, when compared with HbPLT1. Other treatments, especially mechanical wounding, strongly induced HbPLT2 transcripts. These data were consistent with the presence of ethylene and a wound-responsive regulatory cis-element on the sequence of the HbPLT2 promoter. All these findings together with those recently obtained for sucrose transporters and aquaporins are discussed in relation to the different roles for quebrachitol in Hevea brasiliensis.
Assuntos
Euphorbiaceae/genética , Proteínas de Plantas/metabolismo , Polímeros/metabolismo , Sequência de Aminoácidos , Transporte Biológico , Clonagem Molecular , DNA Complementar/genética , Genes de Plantas , Hibridização In Situ , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
Hevea brasiliensis is an important industrial crop for natural rubber production. Latex biosynthesis occurs in the cytoplasm of highly specialized latex cells and requires sucrose as the unique precursor. Ethylene stimulation of latex production results in high sugar flow from the surrounding cells of inner bark towards the latex cells. The aim of this work was to understand the role of seven sucrose transporters (HbSUTs) and one hexose transporter (HbHXT1) in this process. Two Hevea clones were used: PB217 and PB260, respectively described as high and low yielding clones. The expression pattern of these sugar transporters (HbSUTs and HbHXT1) was monitored under different physiological conditions and found to be maximal in latex cells. HbSUT1, one of the most abundant isoforms, displayed the greatest response to ethylene treatment. In clone PB217, ethylene treatment led to a higher accumulation of HbSUT1B in latex cells than in the inner bark tissues. Conversely, stronger expression of HbSUT1B was observed in inner bark tissues than in latex cells of PB260. A positive correlation with HbSUT1B transcript accumulation and increased latex production was further supported by its lower expression in latex cells of the virgin clone PB217.
Assuntos
Etilenos/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Hevea/efeitos dos fármacos , Látex/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Compostos Organofosforados/farmacologia , Proteínas de Plantas/metabolismo , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , DNA de Plantas/genética , Hevea/genética , Hevea/metabolismo , Proteínas de Membrana Transportadoras/genética , Filogenia , Casca de Planta , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/genética , Caules de Planta , Transporte Proteico , Fatores de TempoRESUMO
Natural rubber is synthesized in specialized articulated cells (laticifers) located in the inner liber of Hevea brasiliensis. Upon bark tapping, the laticifer cytoplasm (latex) is expelled due to liber tissue turgor pressure. In mature virgin (untapped) trees, short-term kinetic studies confirmed that ethylene, the rubber yield stimulant used worldwide, increased latex yield, with a concomitant decrease in latex total solid content, probably through water influx in the laticifers. As the mature laticifers are devoid of plasmodesmata, the rapid water exchanges with surrounding liber cells probably occur via the aquaporin pathway. Two full-length aquaporin cDNAs (HbPIP2;1 and HbTIP1;1, for plasma membrane intrinsic protein and tonoplast intrinsic protein, respectively) were cloned and characterized. The higher efficiency of HbPIP2;1 than HbTIP1;1 in increasing plasmalemma water conductance was verified in Xenopus laevis oocytes. HbPIP2;1 was insensitive to HgCl(2). In situ hybridization demonstrated that HbPIP2;1 was expressed in all liber tissues in the young stem, including the laticifers. HbPIP2;1 was up-regulated in both liber tissues and laticifers, whereas HbTIP1;1 was down-regulated in liber tissues but up-regulated in laticifers in response to bark Ethrel treatment. Ethylene-induced HbPIP2;1 up-regulation was confirmed by western-blot analysis. The promoter sequences of both genes were cloned and found to harbor, among many others, ethylene-responsive and other chemical-responsive (auxin, copper, and sulfur) elements known to increase latex yield. Increase in latex yield in response to ethylene was emphasized to be linked with water circulation between the laticifers and their surrounding tissues as well as with the probable maintenance of liber tissue turgor, which together favor prolongation of latex flow.
Assuntos
Aquaporinas/metabolismo , Etilenos/farmacologia , Hevea/citologia , Hevea/metabolismo , Látex/biossíntese , Proteínas de Plantas/metabolismo , Água/fisiologia , Sequência de Aminoácidos , Animais , Aquaporinas/química , Aquaporinas/genética , Sequência de Bases , Western Blotting , Clonagem Molecular , Biologia Computacional , DNA Complementar/genética , Etiquetas de Sequências Expressas , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Hevea/efeitos dos fármacos , Hevea/genética , Cinética , Dados de Sequência Molecular , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Especificidade de Órgãos/efeitos dos fármacos , Casca de Planta/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , XenopusRESUMO
BACKGROUND AND AIMS: The major economic product of Hevea brasiliensis is a rubber-containing cytoplasm (latex), which flows out of laticifers (latex cells) when the bark is tapped. The latex yield is stimulated by ethylene. Sucrose, the unique precursor of rubber synthesis, must cross the plasma membrane through specific sucrose transporters before being metabolized in the laticifers. The relative importance of sucrose transporters in determining latex yield is unknown. Here, the effects of ethylene (by application of Ethrel on sucrose transporter gene expression in the inner bark tissues and latex cells of H. brasiliensis are described. METHODS: Experiments, including cloning sucrose transporters, real time RT-PCR and in situ hybridization, were carried out on virgin (untapped) trees, treated or untreated with the latex yield stimulant Ethrel. KEY RESULTS: Seven putative full-length cDNAs of sucrose transporters were cloned from a latex-specific cDNA library. These transporters belong to all SUT (sucrose transporter) groups and differ by their basal gene expression in latex and inner soft bark, with a predominance of HbSUT1A and HbSUT1B. Of these sucrose transporters, only HbSUT1A and HbSUT2A were distinctly increased by ethylene. Moreover, this increase was shown to be specific to laticifers and to ethylene application. CONCLUSION: The data and all previous information on sucrose transport show that HbSUT1A and HbSUT2A are related to the increase in sucrose import into laticifers, required for the stimulation of latex yield by ethylene in virgin trees.
Assuntos
Etilenos/farmacologia , Hevea/citologia , Hevea/metabolismo , Látex/biossíntese , Sacarose/metabolismo , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Transporte Biológico/efeitos dos fármacos , Clonagem Molecular , DNA Complementar/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glutamato-Amônia Ligase/genética , Glutamato-Amônia Ligase/metabolismo , Hevea/enzimologia , Hevea/genética , Hibridização In Situ , Látex/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Dados de Sequência Molecular , Especificidade de Órgãos/efeitos dos fármacos , Filogenia , Casca de Planta/efeitos dos fármacos , Casca de Planta/genética , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Árvores/efeitos dos fármacos , Árvores/genéticaRESUMO
The latex of Hevea brasiliensis, expelled upon bark tapping, is the cytoplasm of anastomosed latex cells in the inner bark of the rubber tree. Latex regeneration between two tappings is one of the major limiting factors of rubber yield. Hevea species contain high amounts of cyanogenic glucosides from which cyanide is released when the plant is damaged providing an efficient defense mechanism against herbivores. In H. brasiliensis, the cyanogenic glucosides mainly consist of the monoglucoside linamarin (synthesized in the leaves), and its diglucoside transport-form, linustatin. Variations in leaf cyanide potential (CNp) were studied using various parameters. Results showed that the younger the leaf, the higher the CNp. Leaf CNp greatly decreased when leaves were directly exposed to sunlight. These results allowed us to determine the best leaf sampling conditions for the comparison of leaf CNp. Under these conditions, leaf CNp was found to vary from less than 25 mM to more than 60 mM. The rubber clones containing the highest leaf CNp were those with the highest yield potential. In mature virgin trees, the CNp of the trunk inner bark was shown to be proportional to leaf CNp and to decrease on tapping. However, the latex itself exhibited very low (if any) CNp, while harboring all the enzymes (beta-D-diglucosidase, linamarase and beta-cyanoalanine synthase) necessary to metabolize cyanogenic glucosides to generate non-cyanogenic compounds, such as asparagine. This suggests that in the rubber tree bark, cyanogenic glucosides may be a source of buffering nitrogen and glucose, thereby contributing to latex regeneration/production.